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US4472381A - Amino acid derivatives, methods of preparing said derivatives and antihypertensive drugs containing them - Google Patents

Amino acid derivatives, methods of preparing said derivatives and antihypertensive drugs containing them Download PDF

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US4472381A
US4472381A US06/458,844 US45884483A US4472381A US 4472381 A US4472381 A US 4472381A US 45884483 A US45884483 A US 45884483A US 4472381 A US4472381 A US 4472381A
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proline
solution
mole
alanyl
prolyl
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Shumpei Sakakibara
Yasumi Yugari
Shigebumi Hashimoto
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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Priority claimed from JP57141831A external-priority patent/JPS5931788A/ja
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Assigned to AJINOMOTO COMPANY INCORPORATED reassignment AJINOMOTO COMPANY INCORPORATED ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: HASHIMOTO, SHIGEBUMI, SAKAKIBARA, SHUMPEI, YUGARI, YASUMI
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/80—Antihypertensive peptides

Definitions

  • the present invention relates to novel amino acid derivatives and antihypertensive drugs containing them.
  • the present invention also relates to methods of preparing these derivatives.
  • the present inventors have succeeded in the synthesis of novel amino acid derivatives represented by the general formula: ##STR2## Moreover, the present inventors have found that these derivatives, having an antihypertensive activity, are useful as antihypertensive drugs and intermediates for their production, and, based on this finding, have completed the present invention.
  • R 1 and R 2 in the foregoing formula being the same or different from each other, each individually represents hydroxyl, alkyloxy, aryloxy, aralkyloxy, alkyl, aryl, or aralkyl.
  • the alkyloxy is a lower alkyloxy having 1 ⁇ 5 carbon atoms such as methoxy, ethoxy, n-propyloxy, n-butyloxy, and n-pentyloxy.
  • the aryloxy is the one having 6 ⁇ 12 carbon atoms such as phenoxy, naphthyloxy, and p-tolyloxy.
  • the aralkyloxy is the one having 6 ⁇ 12 carbon atoms such as benzyloxy, phenetyloxy, phenylpropyloxy, and hydroxybenzyloxy.
  • the alkyl is a lower alkyl having 1 ⁇ 5 carbon atoms such as methyl, ethyl, n-propyl, n-butyl and n-pentyl.
  • the aryl is the one having 6 ⁇ 12 carbon atoms such as phenyl and p-tolyl.
  • the aralkyl is the one having 6 ⁇ 12 carbon atoms such as benzyl, phenethyl, and phenylpropyl.
  • R 3 , R 5 , R 8 , and R 11 at least two of which are the same or all of which are different from one another, each individually represents hydrogen atom, or alkyl: for example, the one having 1 ⁇ 5 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl.
  • R 3 , R 5 , R 8 , and R 11 may each individually represent hydrogen atom or methyl, or R 3 and R 5 may each individually represent hydrogen atom, and R 8 and R 11 may individually represents methyl; all of them may be different from one another: for example, R 3 represents hydrogen atom, R 5 represents methyl, R 8 represents ethyl, and R 11 represents isopropyl.
  • R 4 , R 7 , and R 10 at least two of which are the same or all of which are different from one another, each individually represents hydrogen atom, or substituted or unsubstituted alkyl, aryl, or aralkyl.
  • the alkyl represents a lower alkyl having 1 ⁇ 5 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and n-pentyl.
  • the aryl represents the one having 6 ⁇ 12 carbon atoms such as phenyl, p-tolyl, and naphthyl.
  • the aralkyl represents the one having 6 ⁇ 12 carbon atoms such as benzyl and naphthylmethyl.
  • Substituent groups in the foregoing substituted groups may include, for example, hydroxyl, carboxyl, carbamoyl, amino guanidino, imidazolyl, indolyl mercapto, and lower alkylthio.
  • R 6 and R 9 being the same or different from each other, each individually represents hydrogen, alkyl, aryl, or aralkyl.
  • the alkyl, the aryl, and the aralkyl being, for example, the foregoing ones, may further include alicylic compounds such as cyclopropyl, cyclopentyl, and cyclohexyl; and aromatic ring-condensed alicyclic compounds such as 2-indanyl and 1-indanyl.
  • R 12 represents hydroxyl, alkyloxy, aryloxy, aralkyloxy, amino, mono- or di-alkyl-, aryl-, or aralkylamino.
  • the alkyloxy, the aryloxy, the aralkyloxy, the alkyl, the aryl, and the aralkyl may each individually include, for example, those cited above as examples of R 1 and R 2 .
  • R 6 and R 7 may form an alkylene bridge having 2 ⁇ 4 carbon atoms, an alkylene bridge having 2 ⁇ 3 carbon atoms and 1 sulfur atom, an alkylene bridge having 3 ⁇ 4 carbon atoms which contains a double bond, or substituted ones of these bridges;
  • R 9 and R 10 may form the same alkylene bridges.
  • substituents on the foregoing bridges include hydroxyl, lower alkoxy, lower alkyl, oxo(o ⁇ ), amino, condensed allyl, a condensed aromatic ring, and a condensed alicyclic ring.
  • Structures formed by such alkylene bridges may be, for example, the following ones: ##STR3##
  • Amino acids constituting the amino acid derivatives of the present invention may be either the L-isomer or the D-isomer.
  • the amino acid derivatives of the present invention may be in the form of a salt, such as a metal salt, as for example, sodium, potassium, lithium, and calcium salts or a salt with an organic base.
  • a salt such as a metal salt, as for example, sodium, potassium, lithium, and calcium salts or a salt with an organic base.
  • organic base there can be adopted amines such as ammonia (ammonium salt), dicyclohexylamine, and N-methyl-D-glucamine; and basic amino acids such as lysine and arginine.
  • amino acid derivatives When the amino acid derivatives are to be included in the antihypertensive drugs of the present invention, they need to be in a form of a pharmaceutically acceptable salt.
  • the amino acid derivatives of the present invention are tripeptide derivatives of which the terminal amino group is combined with a phosphorus compound (P), being represented by the formula: P-X-Y-Z (X, Y, and Z each represents an amino acid residue).
  • P phosphorus compound
  • the amino acid corresponding to X typically includes alanine, leucine, isoleucine, glycine, phenylalanine, arginine, glutamic acid, glutamin, lysine, valine, ornithine, methionine, serine, and threonine;
  • the amino acid corresponding to Y typically includes proline, hydroproline, glycine, N-substituted glycine, and thioproline;
  • the amino acid corresponding to Z typically includes proline, hydroxyproline, glycine, alanine, serine, aspartic acid, arginine, tyrosine, phenylalanine, valine, leucine, isoleucine, threonine, methionine, glutamic acid, glutamine, lysine, cystine, tryptophan, and histidine.
  • the functional groups may be protected by protecting groups ordinarily used in synthetic chemistry of peptides; the protected derivatives are also encompassed by the amino acid derivatives of the present invention.
  • the desired compound can be prepared by causing a phosphoridate [III] such as phosphochloridatediester and phosphochloridatemonoester to react with, for example, a hydrochloride derivative of ester or amide of the tripeptide derivative [II] in an inert solvent such as methylene chloride in the presence of a base such as triethylamine to transform the N-terminal of an the tripeptide derivative into a phosphorus derivative, followed, if necessary, by selectively removing protecting groups by means of hydrolysis with an alkali, catalytic hydrogenation, or the like.
  • a phosphoridate [III] such as phosphochloridatediester and phosphochloridatemonoester
  • an inert solvent such as methylene chloride
  • a base such as triethylamine
  • the desired compound can also be prepared by directly condensing a phosphoridate [III] with a free compound of the tripeptide derivative [II].
  • the desired compound can be prepared by subjecting a phosphorus derivative of a dipeptide [IV] and an amino acid derivative [V] such as a hydrochloride of ester or amide to a condensation reaction in an inert solvent such as dimethylformamide (DMF) with the use of a condensing agent such as N,N'-dimethylaminopropylethylcarbodiimide (WSC) and 1-hydroxybenzotriazole (HOBt) to obtain a phosphorus derivative of the tripeptide [I], followed, if necessary, by selectively removing protecting groups by means of hydrolysis with an alkali or catalytic hydrogenation.
  • a condensing agent such as N,N'-dimethylaminopropylethylcarbodiimide (WSC) and 1-hydroxybenzotriazole (HOBt)
  • the desired compound can be obtained by subjecting a phosphorus derivative of an amino acid [VI] and a dipeptide derivative [VII] such as a hydrochloride of ester or amide to a condensation reaction in an inert solvent such as dimethylformamide with the use of a peptide condensating agent used in synthetic chemistry of peptides, such as WSC and HOBt, to obtain a phosphorus derivative of a tripeptide, followed, if necessary, by selectively removing protecting groups by means of hydrolysis with an alkali or catalytic hydrogenation.
  • a phosphorus derivative of an amino acid [VI] and a dipeptide derivative [VII] such as a hydrochloride of ester or amide
  • an inert solvent such as dimethylformamide
  • a substituted phosphoryl derivative can be prepared by preparing a peptide derivative represented by the general formula: ##STR7## wherein Z represents the same as defined above, followed by causing the peptide derivative to react with a substituted phosphoryl halogenide such as diethyl- or dibenzylphosphoryl chloride.
  • the substituted phosphoryl derivative for example, dibenzylphosphoryl chloride, can be converted into a phosphoryl derivative by subjecting it to hydrogenation using palladium carbon as catalyst.
  • the foregoing tripeptide an intermediate for the derivatives of the present invention, can be prepared by causing an alanine having a protected amino group to react with a proline having a protected carboxyl group to prepare alanylproline, removing the protecting groups of the alanylproline, and causing the resulting product to react with the Z component, an amino acid having a protected amino group, followed, if necessary, by removing the protecting group.
  • the tripeptide can also be prepared by preparing dibenzylphosphorylalanylproline, follwed by causing the product to react with the foregoing selected amino acid ester according to the method of Japanese patent first publication No. 104863/1981.
  • alanylproline derivative of which the constituent proline residue has a hydroxyl group (hydroxyproline) can also be prepared in the same manner.
  • Those protecting groups for amino, imino, carboxyl, and hydroxyl groups those methods for the protection, those methods for removal of the protecting groups, and those methods for amide-linking by condensation between amino and carboxyl groups which are all employed in the preparation of the derivatives of the present invention and intermediates therefore may be the ones ordinarily used in methods for peptide synthesis or generally or conventionally employed in the known literature, for example, Protein Chemistry, 1 Amino Acid.Peptide, e.g. pages 405 ⁇ 509 (1969), compiled by Shiro Akabori, Takeo Kaneko, and Kozo Narita, published by Kyoritsu Shuppan Co.
  • An amide-linkage is advantageously formed from an amino acid by a condensation method in which an active ester of the amino acid having a protected amino group, such as p-nitrophenylester and N-hydroxysuccinimideester, is used for reaction.
  • an active ester of the amino acid having a protected amino group such as p-nitrophenylester and N-hydroxysuccinimideester
  • DMF or water can be adopted as solvent.
  • the reaction temperature may be a room temperature or so, but the reaction can also, as needed, be accelerated by heating.
  • the derivatives of the present invention are isolated from the reaction mixture by concentrating the reaction mixture to dryness, purifying the residue by means of column chromatography, followed by lyophilization of the product.
  • an antihypertensive drug when used, as an active ingredient, for an antihypertensive drug, there may be adopted their free forms, their nontoxic forms of salt, or their nontoxic forms having protecting groups.
  • An amino acid constituting the derivatives for use as an antihypertensive drug of the present invention may be either the L-isomer or the D-isomer.
  • the amino acid derivatives of the present invention are useful as an antihypertensive drug for treating hypertensive mammals including humans.
  • the derivatives can be used for lowering blood pressure by formulating them into a preparation such as tablets, capsules, and elixirs for oral administration and into an aspetic liquid preparation or an aseptic suspension preparation for parenteral administration.
  • the amino acid derivatives of the present invention can be administered to a subject necessitating such treatment (animals and humans) in a dosage range of 0.2 ⁇ 500 mg per subject generally several times a day, that is, in a total daily dosage of 1 ⁇ 2000 mg.
  • the dosage varies according to the seriousness of disease, the body weight of subjects, and other factors acknowledged by those skilled in the art.
  • amino acid derivatives of the present invention can also be administered together with diuretics or other antihypertensive drugs.
  • these drugs are administered in a dosage combination of which one unit of daily dose is in the range from 7/8 times as large as a clinical dosage minimally recommended, to a level maximally recommended singly for each entity of disease.
  • a kind of antihypertensive drugs of the present invention which is clinically effective in a daily dosage range of 15 ⁇ 200 mg can effectively be administered together with the following other antihypertensive drugs and diuretics in a daily dosage range of 3 ⁇ 200 mg: hydrochlorothiazide (15 ⁇ 200 mg), chlorothiazide (125 ⁇ 2000 mg), ethacrynic acid (15 ⁇ 200 mg), amiloride (5 ⁇ 20 mg), furosemide (5 ⁇ 80 mg), propranolol (20 ⁇ 480 mg), timolol (5 ⁇ 50 mg), methyldopa (65 ⁇ 2000 mg).
  • the foregoing dosage ranges are adjusted on the basis of unit according to the necessity for the possible daily divided dosage. The dosage varies according to the seriousness of disease, the body weight of subject, and other factors acknowledged by those skilled in the art.
  • compositions stated below The foregoing typical combinations of drugs are formulated into pharmaceutical compositions stated below.
  • About 0.2 ⁇ 500 mg of the derivatives of the present invention, pharmaceutically acceptable salt compounds, or mixtures of both are blended into unit dosage forms generally acknowledged or required for the pharmaceutical practice together with pharmaceutically acceptable vehicles, carriers, excipients, binders, antiseptics, stabilizers, flavorings, and so forth.
  • the amount of each active substance in these compositions or preparations is adjusted in such a way as to give an appropriate dosage of the prescribed range.
  • a binder such as traganth, gum arabic, cornstarch, and gelatin; an excipient such as microcrystalline cellulose; a swelling agent such as cornstarch, pregelatinized starch, and arginic acid; a lubricant such as magnesium stearate; a sweetner such as sucrose, lactose, and saccharin; a flavoring such as peppermint, an oil from Gaultheria adenothrix Maxim, and cherry.
  • a liquid carrier such as fatty oil can further be incorporated in the foregoing type materials.
  • tablets can be coated with shellac and/or sugar.
  • Syrups or elixirs can contain active compounds, sucrose as a sweetner, methyl- and propylparaben as an antiseptic, a coloring matter, a flavoring such as cherry and an orange flavoring.
  • Aseptic compositions for injection can be formulated according to the usual practice for preparation of pharmaceutical dosage forms, in which practice an active substance is dissolved or suspended in a vehicle such as water for injection; natural vegetable oils such as sesame oil, palm oil, peanut oil, and cotton seed oil; and synthetic fat vehicle such as ethyl oleate.
  • a vehicle such as water for injection; natural vegetable oils such as sesame oil, palm oil, peanut oil, and cotton seed oil; and synthetic fat vehicle such as ethyl oleate.
  • a buffer, an antiseptic, and an antioxidant can further be incorporated as occasion demands.
  • a phosphorus trichloride (3.7 g, 25 m mole) in benzene (15 ml) solution was added dropwise and slowly, to the mixture of dimethylaniline (6.1 g, 50 m mole) and benzylalcohol (5.4 g, 50 m mole) at a temperature of less than 15° C. by cooling with coolant of -15° C. and stirring. After the addition, the mixture was further stirred for 30 minutes. To the mixture, benzylalcohol (2.7 g, 25 m mole) was further added dropwise and the mixture was reacted at a room temperature overnight.
  • reaction solution was shaken with water (15 ml), and the thus obtained organic phase was separated and then washed with water, 5N aqueous ammonia and water in this order.
  • the thus obtained mixture was dried over anhydrous sodium sulfate.
  • the drying agent was removed by filtration and the remaining filtrate was distilled under reduced pressure leaving a non-coloured and oily dibenzylhalogen phosphite product (3.2 g).
  • Dibenzyl hyddrogen phosphite (6.7 g) was dissolved in carbon tetrachloride (70 ml) dried previously, and put in a 3-neck flask.
  • Sulfurylchloride (3 g) in carbon tetrachloride (10 ml) solution was added dropwise to the above mixture at a temperature of less than 10° C. while cooling with coolant of -15° C. and stirring and at an atmosphere of nitrogen gas.
  • N-t-butyloxycarbonyl-L-alanine (3.8 g, 20 m mole), L-proline benzyl ester hydrochloride (5 g, 20.6 m mole) and 1-hydroxy benzotriazole, which is hereinafter referred to as HOBt (2.7 g, 20 m mole) were suspended in tetrahydrofuran, which is hereinafter referred to as THF (50 ml).
  • THF tetrahydrofuran
  • N,N'-dimethylaminopropylethylcarbodiimide, which is hereinafter referred to as WSC (3.8 ml) in THF (10 ml) solution was added dropwisely to the above suspended solution while cooling a -15° C. and stirring.
  • WSC (2.8 ml) was added dropwise to the above mixture while cooling to -15° C. and stirring. The reaction was carried out for 3 hours at a temperature of not more than 0° C., and then overnight at room temperature. The solvent was removed by distillation under reduced pressure leaving a residue.
  • N-t-butyloxycarbonyl-L-alanyl-L-prolyl-L-proline benzyl ester (6.0 g, 12.7 m mole) was dissolved in 4.8N hydrogen chloride in dioxan (15 ml) under shaking and stirred for 40 minutes at room temperature. The solvent was distilled off under reduced pressure and dried ethyl ester was added thereto. Thus obtained precipitate was obtained on the paper filter and soon put into the desiccator containing sodium hydroxide to be dried. All amount of the precipitate was dissolved in DMF (30 ml) and cooled to a temperature of -5° C. Triethyl amine was added to the cold mixture to neutralize it.
  • the oily material obtained above was dissolved in a mixture (20 ml) of acetone and methanol (1:1), and 1N sodium hydroxide (15 ml) was added thereto under cooling with ice-bath. The mixture was stirred for 1.5 hours at room temperature and was neutralized with 1N hydrochloric acid. The organic solvent was distilled off under reduced pressure. The residue was dissolved in ethyl acetate and washed with 1N hydrochloric acid and water, and dried with anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline (5.7 g).
  • the oily material (660 mg) was dissolved in ethanol (5 ml) and an aqueous L-arginine solution (1.2 ml) was added thereto.
  • the arginine solution has been prepared by dissolving L-arginine in the water in a ratio of 1.74 g of L-argine in 10 ml of the solution.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole) as shown in the Japanese patent first publication No. 104863/1981, HOBt (338 mg, 2.5 m mole) and L-serine methylester hydrochloride (467 mg, 3 m mole) were suspended in DMF (10 ml), and WSC (0.5 ml) was added dropwise thereto while cooling to -15° C. and stirring. The reaction was carried out for 3 hours under cooling and next overnight at a room temperature. Ethyl acetate (100 ml) was added to the reaction solution, washed 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate.
  • the solution was distilled under reduced pressure to eliminate the organic solvent, and the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate.
  • the thus obtained solution was distilled under reduced pressure to give oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-serine (1.2 g).
  • the oily material (380 mg) was dissolved in ethanol (5 ml) and the aqueous L-arginine solution (0.71 ml) as prepared above.
  • the solution was distilled under the reduced pressure leaving a residue, and residual water was removed completely by azeotropic distillation with toluene.
  • methanol was added and an insoluble material was separated by filtration.
  • the filtrate was distilled under reduced pressure to give an amorphous powder of dibenzylphosphoryl-L-alanyl-L-prolyl-L-serine L-arginine salt (230 mg).
  • a fixed weight of the sample was heated in 6N hydrochloric acid at 110° C. for 19 hours. From this hydrolized product, amino acids analysis was carried out. The ratio of alanine, proline, serine and arginine was 1.00:1.02:0.96:1.07.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg, 2.5 m mole) and L-aspartic acid dibenzylester P-toluene sulfonic acid salt (1.45 g, 3 m mole) were suspended in DMF (10 ml), and WSC (0.5 ml) was added dropwise thereto while cooling at -15° C. and stirring. The reaction was carried out under cooling for 3 hours and thereafter at room temperature overnight. To the reaction solution ethyl acetate (100 ml) was added and thus obtained mixture was washed with 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate.
  • oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-asparatic acid dibenzylester (1.9 g).
  • This oily material was dissolved in a mixture (20 ml) of acetone and methanol (1:1 ), and 1N sodium hydroxide (5 ml) was added thereto while cooling.
  • the oily material (430 mg) was dissolved in ethanol (5 ml), and aqueous arginine solution (0.76 ml), which has been prepared by dissolving L-arginine in water in the ratio of 1.74 g of L-arginine per 10 ml of the aqueous arginine solution, was added thereto.
  • the solvent was removed by distillation under reduced pressure, and then the water was removed completely by azeotropic distillation with toluene. Methanol was added to the residue, and thus prepared insoluble material was separated by filtration.
  • the solvent was distilled off under reduced pressure to obtain amorphous powder of dibenzylphosphoryl-L-alanyl-L-prolyl-L-aspartic acid L-arginine salt (390 mg).
  • the fixed weight of the sample was heated in 6N hydrochloric acid at 110° C. for 19 hours.
  • amino acids analysis was carried out.
  • the ratio of alanine, proline, aspartic acid and arginine was 1.00:0.97:1.04:1.04.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg, 2.5 m mole) and L-tyrosine ethylester hydrochloride (737 mg, 3 m mole) was suspended in DMF (10 ml), and WSC (0.5 ml) was added gradually thereto while cooling to -15° C. and stirring. The reaction was carried out for 3 hours under cooling and then overnight at room temperature. To the reaction solution ethyl acetate (100 ml) was added, and the mixture was washed with 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate.
  • the oily material was dissolved in a mixture of acetone and methanol (1:1), and 1N sodium hydroxide (5 ml) was added thereto while cooling. Next, the mixture was stirred for 1.5 hours at room temperature, and neutralized with 1N hydrochloric acid. The thus obtained solution was distilled under reduced pressure leaving a residue and the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid and water and dried with anhydrous sodium sulfate. The thus dried solution was distilled under reduced pressure to obtain an oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-tyrosine material (1.2 g).
  • a fixed weight of the sample (the product) was heated in 6N hydrochloric acid for 19 hours at 110° C., and amino acids in thus hydrolized material were analyzed.
  • the ratio of alanine, proline, tyrosine and arginine was 1.00:0.99:1.03:1.02.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg, 2.5 m mole) and L-phenylalanine ethylester hydrochloride (690 mg, 3 m mole) were suspended in DMF (10 ml) and WSC (0.5 ml) were added thereto while cooling to -15° C. and stirring. The reaction was carried out for 3 hours under cooling and then overnight at room temperature. To the reaction solution, ethyl acetate (100 ml) was added, and the mixture was washed with 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate.
  • the the dried solution was distilled under reduced pressure to obtain an oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-phenylalanine ethylester material (1.3 g).
  • the oily material was dissolved in a mixture (20 ml) of acetone and methanol (1:1) and 1N sodium hydroxide (5 ml) was added under cooling. The mixture was stirred for 1.5 hours at room temperature, and neutralized with 1N hydrochloric acid. The thus neutralized solution was distilled under reduced pressure leaving a residue and the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid and water, and dried with anhydrous sodium sulfate. The thus dried solution was distilled under reduced pressure leaving an oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-phenylalanine (0.9 g).
  • the oily material (280 mg) was dissolved in ethanol (5 ml), and the aqueous arginine solution (0.47 ml) as previously described was added thereto.
  • the hydrochloric solution was distilled under reduced pressure leaving a residue and water was completely removed by azeotropic distillation with toluene.
  • methanol was added and the thus prepared insoluble matter was separated by filtration.
  • the filtrate was distilled under reduced pressure leaving a dibenzylphosphoryl-L-alanyl-L-prolyl-L-phenylalanine L-arginine salt material (270 mg).
  • the fixed weight of the sample was heated in 6N hydrochloric acid for 19 hours at 110° C. In the thus hydrolized material amino acids were analyzed.
  • the ratio of alanine, proline, phenylalanine and arginine was 1.00:1.00:1.08:1.05.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg, 2.5 m mole) and glycine benzylester p-toluene sulfonic acid salt (1.0 g, 3 m mole) were suspended in DMF (10 ml), and WSC (0.5 ml) was dropwise added thereto while cooling to -15° C. and stirring. The reaction was carried out for 3 hours under cooling, and next, overnight at room temperature. To the reaction solution ethyl acetate (100 ml) was added and the mixture was washed with 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate.
  • This oily material was dissolved in a mixture (20 ml) of acetone and methanol (1:1) and 1N sodium hydroxide (5 ml) was added thereto under cooling. Next, the mixture was stirred for 1.5 hours at room temperature, and neutralized with 1N hydrochloric acid. The thus neutralized solution was distilled under reduced pressure leaving a residue, and the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate. The thus dried solution was distilled under reduced pressure leaving an oily dibenzylphosphoryl-L-alanyl-L-prolylglycine material (1.4 g).
  • the oily material (610 mg) was dissolved in ethanol (5 ml) and the aqueous arginine solution as previously described (1.2 ml) was added thereto.
  • the hydrochloric solution was distilled under reduced pressure, and further water was removed completely by azeotropic distillation with toluene leaving a residue.
  • the thus obtained filtrate was distilled under reduced pressure to obtain an amorphous powder of dibenzylphosphoryl-L-alanyl-L-prolyl-glycine L-arginine salt material (570 mg).
  • the fixed weight of the sample was heated in 6N hydrochloric acid for 19 hours at 110° C.
  • amino acids were analyzed.
  • the ratio of alanine, proline, glycine and arginine was 1.00:0.91:1.00:1.11.
  • Dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt (3 g, 4.2 m mole), and L-arginine (1.45 g, 8.36 m mole) were dissolved in a mixture of ethanol (50 ml) and water (10 ml), and palladium-carbon (1 g) was added thereto. After that catalytic reduction reaction was carried out for 5 hours. The catalyst was removed by filtration and the solvent was removed by distillation. The residue was dissolved in water and freeze-dried to obtain phosphoryl-L-alanyl-L-prolyl-L-proline tri-L-arginine salt (3.63 g). After hydrolysis of the material with 6N hydrochloric acid, amino acids were analyzed. The ratio of alanine, proline and arginine was 1.00:1.99:2.90.
  • Dibenzylphosphoryl-L-alanyl-L-proline (2.8 g, 6.4 m mole), HOBt (952 mg, 7 m mole), and L-proline amide (805 mg, 7 m mole) were dissolved in DMF (10 ml), and N,N'-dimethylaminopropylethylcarbodiimide hydrochloride (1.34 g, 7 m mole) was added thereto while cooling to -15° C.
  • oily material was dissolved in acetone (25 ml), and 1N sodium hydroxide (4 ml) was added thereto while cooling with ice. Next, the mixture was stirred for 1 hour at room temperature and neutralized with 1N hydrochloric acid. The thus neutralized solution was distilled under reduced pressure, and the residue was dissolved in ethyl acetate. The mixture was washed with 1N hydrochloric acid and water in order, and dried anhydrous sodium sulfate. The solution was distilled under reduced pressure, and the thus obtained oily material was dissolved in ethanol (3 ml). The aqueous arginine solution (1.2 ml) as described above, was added thereto, and the aqueous solution was distilled under reduced pressure.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.8 g, 4 m mole), HOBt (540 mg), and L-arginine methylester 2 hydrochloric acid salt (2.62 g, 10 m mole) were dissolved in DMF (30 ml), and then triethylamine (2.8 ml, 20 m mole) and dicyclohexylcarbodiimide (1.03 g) were added thereto while cooling to -15° C. The reaction was carried out for 4 hours under cooling and then overnight at room temperature. Thus produced dicyclohexylurea was separated by filtration. The solvent was distilled off and 0.1N hydrochloric acid was added to the residue.
  • the mixture was washed with ethyl ether and the aqueous layer was absorbed with ion exchanging resin "Dia ion HP-20" as produced by Mitsubishikaseikogyo Co., Inc.
  • the eluants were concentrated and the thus obtained residue was dissolved in acetone (10 ml).
  • 0.1N sodium hydroxide (5 ml) was added to the mixture under cooling and the mixture was stirred for 1 hour at room temperature.
  • the mixture was neutralized with 1N hydrochloric acid and the thus neutralized solution was distilled under reduced pressure leaving a residue.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.4 g, 3.2 m mole), HOBt (476 mg, 3.5 m mole) and D-alanine benzylester p-toluene sulfonic acid salt (1.3 g, 3.5 m mole) were dissolved in DMF (5 ml), and WSC (0.64 ml) was added thereto while cooling to -15° C. The reaction was carried out for 3 hours under cooling and then overnight at room temperature. To the reaction solution ethyl acetate (100 ml) was added and the mixture was washed with 1N hydrochloric acid, 5% sodium bicarbonate and water in order. The mixture was dried with anhydrous sodium sulfate and the thus obtained solution was distilled under reduced pressure to give an oily dibenzylphosphoryl-L-alanyl-L-prolyl-D-alanine benzylester material.
  • Dibenzylphosphoryl-L-alanyl-L-proline (1.4 g, 3.2 m mole), HOBt (476 mg, 3.5 m mole), and trans-L-hydroxyproline benzylester p-toluene sulfonic acid salt (1.4 g, 3.5 m mole) were dissolved in DMF, and WSC (0.64 ml, 3.5 m mole) was added thereto while cooling to -15° C. The reaction was carried out for 3 hours under cooling and then overnight at room temperature.
  • oily material was dissolved in acetone (15 ml), and 1N sodium hydroxide (4 ml) was added thereto under cooling with ice. The mixture was stirred for 2 hours at room temperature and neutralized with 1N hydrochloric acid.
  • the solvent was distilled off under reduced pressure and the residue was dissolved in water, and washed with ethyl acetate.
  • the water solution was treated with ion exchanging resin "Dia ion HP-20" for adsorption of desired product.
  • the eluate was concentrated under reduced pressure and the residue was dissolved in ethanol, and the aqueous arginine solution as mentioned above (1.2 ml) was added thereto.
  • the thus obtained aqueous solution was distilled under reduced pressure and further water was completely removed by azeotropic distillation with toluene.
  • oily material was dissolved in methanol (20 ml) and 1N sodium hydroxide (6 ml) was added thereto while cooling with ice, and stirred for 3 hours at room temperature.
  • the mixture was neutralized with 1N hydrochloric acid, and the solvent was distilled off under reduced pressure.
  • the residue was dissolved in water and treated with ion exchanging resin "Dia ion HP-20" for adsorption of the desired product. The resin was washed with water, and the desired product was eluted with 80% methanol.
  • Trifluoroacetic acid (10 ml) was added to t-butyloxycarbonylglycyl-L-prolyl-L-proline (1.85 g, 5 m mole) while cooling and the mixture was stirred for 40 minutes.
  • the trifluoroacetic acid solution was distilled under reduced pressure, and to the residue, ethyl ether was added. The thus produced precipitate was obtained on the filter paper, and dissolved in DMF (10 ml).
  • the mixture was neutralized by adding triethylamine (1.5 ml) to the mixture while cooling to -5° C., and dibenzylphosphorylchloride carbon tetrachloride solution (7 m mole) and triethylamine were dropwise added thereto while keeping a temperature not more than 5° C., and keeping a pH range to 8-9.
  • the mixture was stirred for 3 hours at room temperature and distilled until a residue was obtained.
  • the thus obtained residue was dissolved in ethyl acetate (100 ml), washed with 1N hydrochloric acid and water in this order, and dried with anhydrous sodium sulfate.
  • reaction solution was usually kept from 8 to 9 by adding triethylamine.
  • the mixture was stirred for 3 hours at room temperature, and then distilled until a residue was obtained.
  • the residue was dissolved in ethyl acetate (100 ml) and washed with 1N hydrochloric acid, 5% sodium bicarbonate and water in order, and dried with anhydrous sodium sulfate.
  • the thus obtained solution was distilled under reduced pressure to obtain an oily dibenzylphosphoryl-L-phenylalanyl-L-prolyl-L-proline benzylester material.
  • the oily material was dissolved in acetone (20 ml) and 1N sodium hydroxide (4 ml) was added thereto while cooling with ice and the mixture was stirred for 3 hours at room temperature. After neutralization of the mixture with 1N hydrochloric acid, the thus neutralized solution was distilled under reduced pressure. The residue was dissolved in ethyl acetate. The mixture was dried with 1N hydrochloric acid and water in order, and dried with anhydrous sodium sulfate. The thus obtained solution was distilled under reduced pressure, and the residue was reprecipitated with ethyl acetate and n-hexan to obtain dibenzylphosphoryl-L-phenylalanyl-L-prolyl-L-proline (820 mg).
  • dibenzylphosphorylchloride carbon tetrachloride solution (3 m mole) and triethylamine were added dropwise while keeping the temperature to not more than 5° C., and keeping usually pH value to 8-9.
  • the mixture was stirred for 3 hours at room temperature, then distilled off under reduced pressure, and the residue was dissolved in water and then passed through "Dowex 1 ⁇ 2" (a type of CH 3 COO - ).
  • the desired product was eluted with water and the thus obtained eluate was freeze-dried.
  • the aqueous phase was acidified with 1N hydrochloric acid, and the desired product was extracted with ethyl acetate.
  • the ethyl acetate phase was washed with sodium chloride solution and dried with anhydrous sodium sulfate.
  • the ethyl acetate was distilled off under reduced pressure. The residue was re-precipitated with ethyl acetate-ethyl ether to obtain dibenzylphosphoryl-L-glutamyl-L-prolyl-L-proline (140 mg).
  • oily material was dissolved in methanol (20 ml) and 1N sodium hydroxide (20 ml) was added thereto under cooling. The mixture was stirred for 3 hours, and the aqueous solution was adjusted to pH 7 by addition of 1N hydrochloric acid. The methanol was distilled off under reduced pressure. The aqueous solution was adjusted to pH 2 by addition of 1N hydrochloric acid, and the desired product was extracted with ethyl acetate. The ethyl acetate phase was washed with water and dried with anhydrous sodium sulfate. The ethyl acetate solution was distilled under reduced pressure leaving an oily t-butyloxycarbonyl- ⁇ -carbobenzoxy-L-lysyl-L-proline.
  • Trifluoroacetic acid 50 ml was added to the oily t-butyloxycarbonyl- ⁇ -carbobenzoxy-L-lysyl-L-prolyl-L-proline benzylester (11 g) under cooling and the mixture was stirred for 40 minutes.
  • Trifluoroacetic acid was distilled off under reduced pressure and the residue was dissolved in DMF (30 ml), and was neutralized by addition of triethylamine, and then diethylphosphorylchloride carbon tetrachloride solution (25 m mole) was added thereto while keeping the temperature to not more than 5° C. and keeping usually pH value to 8-9. The mixture was stirred for 4 hours at room temperature.
  • the solvent was distilled off under reduced pressure and the residue was dissolved in ethyl acetate, and washed with 1N hydrochloric acid, 5% sodium bicarbonate and water in order.
  • the solution was dried with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain an oily diethylphosphoryl- ⁇ -carbobenzoxy-L-lysyl-L-prolyl-L-proline benzylester (9.5 g).
  • the oily material (9.5 g) was dissolved in methanol (100 ml) and palladium-carbon (5 g) was added thereto, and thereafter catalytic reductive reaction was carried out for 4 hours.
  • the catalyst was removed by filtration and the solution was distilled under reduced pressure leaving an oily diethylphosphoryl-L-lysyl-L-prolyl-L-proline residue.
  • the oily material was dissolved in ethanol and L-arginine (2.3 g) water (4 ml) solution was added thereto.
  • the solvent was distilled off under reduced pressure and water was removed by azeotropic distillation with toluene. Ethanol was added to the residue, and the thus produced insoluble material was removed by filtration.
  • diethylphosphoryl-L-lysyl-L-prolyl-L-proline L-arginine salt (3.5 g) was obtained.
  • Trifluoroacetic acid 50 ml was added to t-butyloxycarbonyl-L-alanyl-L-proline benzylester (11.3 g, 30 m mole) under cooling and the mixture was stirred for 40 minutes. Trifluoroacetic acid was distilled off under reduced pressure and the residue was dissolved in DMF and neutralized by addition of triethylamine (8 ml) under cooling to -5° C. Next, diethylphosphorylchroride carbon tetrachloride solution (40 m mole) was added dropwisely thereto while keeping the temperature to not more than 5° C., and keeping pH value to 8-9 and the mixture was stirred overnight at room temperature.
  • the solution was distilled under reduced pressure leaving a residue, and the residue was dissolved in ethyl acetate.
  • the solution was washed with 1N hydrochloric acid, 5% sodium bicarbonate and water in order, and dried with anhydrous sodium sulfate.
  • the solution was distilled under reduced pressure leaving an oily diethylphosphoryl-L-alanyl-L-proline benzylester residue (9.2 g).
  • the oily diethylphosphoryl-L-alanyl-L-proline benzylester (8.5 g, 20.6 m mole) was dissolved in methanol and palladium-carbon (2 g) was added thereto. The reductive reaction was carried out for 3 hours. The catalyst was removed by filtration and the solvent was distilled off under reduced pressure to obtain an oily diethylphosphoryl-L-alanyl-L-proline.
  • oily material was dissolved in methanol, and palladium-carbon (2 g) was added thereto.
  • the reductive reaction was carried out for 3 hours.
  • the catalyst was separated by filtration and the solvent was distilled off under reduced pressure leaving a residue.
  • the oily material was dissolved in ethanol and L-arginine (1.3 g) water (2 ml) solution was added thereto.
  • Dibenzyl hydrogene phosphite (12.3 g, 50 m mole) was dissolved in DMF (80 ml), and sodium hydride (60% in oil, 2.2 g) was added to the mixture at an atmosphere of nitrogen while cooling to -15° C. and stirring. The thus obtained mixture was stirred for 1.5 hours at the temperature not more than 0° C.
  • Phenethyl bromide (9.25 g, 50 m mole) in DMF (10 ml) solution was added dropwise to the above solution at an atmosphere of nitrogen at the temperature not more than 0° C. The thus obtained mixture was further reacted overnight at room temperature.
  • DMF solution was distilled under reduced pressure leaving a residue and the residue was dissolved in ethyl ether.
  • Dibenzylphenethylphosphonate (3.19 g, 8.7 m mole) was dissolved in carbon tetrachloride (5 ml) and phosphorus pentachloride (1.91 g, 9.2 m mole) was added thereto under cooling in the ice bath, and the mixture was stirred for 30 minutes. The mixture was heated to 70° C. in the rate of 10° C. per 15 minutes and stirred for 30 minutes at 70° C. The reaction was carried out to completion for 2 hours at 70° C./1 mmHg in the rotating evaporator, and then the solvent, phosphorus oxychloride and benzyl chloride were distilled off.
  • N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline benzylester (0.88 g, 1.4 m mole) was dissolved in acetone (5 ml) and sodium hydroxide (0.18 g, 4.5 m mole) in water (4 ml) solution was added thereto under cooling in the ice bath, and the mixture was stirred for 2 hours.
  • water (100 ml) was added and the mixture was washed with ethyl ether (50 ml).
  • the solution was neutralized with 1N hydrochloric acid, and the desired product was extracted with ethyl ether (100 ml).
  • the ethyl ether solution was dried with anhydrous sodium sulfate.
  • the thus obtained solution was distilled under reduced pressure yielding an oily N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline material (0.54 g, yield: 71.6%).
  • the thus obtained oily material (1.0 m mole) was dissolved in ethanol (4 ml) and a solution of L-arginine (0.21 g, 1.0 m mole) in water (4 ml) was added thereto.
  • the thus obtained solution was distilled under reduced pressure.
  • N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline L-arginine salt (0.59 g, yield: 60.2% from N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline benzylester) was obtained.
  • N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline benzylester (0.67 g, 1.1 m mole) was dissolved in the mixture (24 ml) of water and methanol (1:1) and sodium bicarbonate (0.16 g, 2.2 m mole) was added thereto. Hydrogen gas was passed through the solution for 3 hours under atmospheric pressure at room temperature in the presence of 5% palladium carbon as a catalyst.
  • the catalyst was separated by filtration and the solution was distilled leaving a residue. By dissolving the residue in water and freeze-drying it, N-phenethylphosphonyl-L-alanyl-L-prolyl-L-proline 2 sodium salt (0.54 g, quantitative yield) was obtained.
  • L-proline benzylester hydrochloride (20 g, 82.7 m mole) was added to chloroform (200 ml) and triethylamine (8.3 g, 82.7 m mole), N-t-butyloxycarbonyl-glycine (14.5 g, 82.7 m mole), and HOBt (11.2 g, 82.7 m mole) were added thereto under cooling.
  • WSC hydrochloride (15.82 g, 82.7 m mole) was added dropwise.
  • reaction was carried out for 2 hours on ice, and next, overnight at room temperature.
  • the reaction solution was washed with 10% citric acid, aqueous sodium chloride, 5% sodium bicarbonate and aqueous sodium chloride in order, and dried with anhydrous sodium sulfate.
  • the solution was distilled enough under reduced pressure leaving a sticky N-t-butyloxycarbonyl-glycyl-L-proline benzylester product (31.4 g, 83.5 m mole; yield: 100%).
  • Glycyl-L-proline benzylester hydrochloride (20.0 g, 64.1 m mole) was dissolved in dried ethanol (150 ml) and indan-2-on (4.23 g, 32.1 m mole), and Molecular Sieves (3A) (5 g) were added thereto.
  • Sodium cyanoborohydride (2.01 g, 32.1 m mole) was gradually added to the above solution while cooling with ice, and stirring. The mixture was stirred for 1 hour under cooling with ice and then stirred for 2 hours at room temperature. A little insoluble material was separated by filtration. The ethanol was removed by distillation under reduced pressure from the reaction solution.
  • N-(2-indanyl) glycyl-L-proline benzylester (6.0 g, 15.9 m mole), N-t-butyloxycarbonyl-L-alanine (3.0 g, 15.9 m mole) and HOBt (2.1 g, 15.9 m mole) were dissolved in chloroform (60 ml) and the mixture was stirred under cooling with ice, and WSC hydrochloride (3.03 g, 15.9 m mole) was added dropwise thereto. The reaction solution was left for 4 days in a refrigerator.
  • chloroform 100 ml was added and the mixture was washed with 10% citric acid, aqueous sodium chloride, 5% sodium bicarbonate and aqueous sodium chloride in this order, and dried with anhydrous sodium sulfate.
  • the solution was distilled under reduced pressure leaving a syrupy residue.
  • the syrupy residue was dissolved in ethyl ether (300 ml), and water (200 ml) was added thereto.
  • the pH value was adjusted to 1.5 with 6N hydrochloric acid and the mixture was washed.
  • the ethyl ether layer was separated and then dried with anhydrous sodium sulfate.
  • N-t-butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline benzylester (2.72 g, 4.95 m mole) was dissolved in 4N hydrogen chloride dioxane solution (50 ml) and the mixture was stirred for 1 hour at room temperature. The solvent was distilled under reduced pressure from the solution, and thereby a sticky residue was obtained.
  • N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl) glycyl-L-proline benzylester (0.74 g, 1.0 m mole) was dissolved in a mixture (30 ml) of water and ethanol (2:3) and sodium bicarbonate (0.176 g, 2.0 m mole) was added thereto. Hydrogen gas was passed through the solution for 3 hours at room temperature and atmospheric pressure in the presence of 5% palladium carbon as a catalyst. The catalyst was separated by filtration and the solution was distilled under reduced pressure until a residue was obtained.
  • N-phenethylphosphonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline 2 sodium salt (0.57 g, yield: 95.4%) was obtained.
  • N-t-Butyloxycarbonyl-glycyl-L-proline methylester L-proline methylester hydrochloride (13.6 g, 82.7 m mole) was suspended in chloroform (200 ml) and to the suspended solution triethylamine (8.3 g, 82.7 m mole) was added while cooling.
  • N-t-butyloxycarbonyl glycine (14.5 g, 82.7 m mole) was added and WSC hydrochloride (15.82 g, 82.7 m mole) was gradually added thereto while cooling with ice. The reaction was carried out for 2 hours while cooling with ice, and next overnight at room temperature.
  • the reaction solution was washed with 10% citric acid, aqueous sodium chloride, 5% sodium bicarbonate and aqueous sodium chloride in order, and dried with anhydrous sodium sulfate.
  • the solution was distilled under reduced pressure leaving a syrupy N-t-butyloxycarbonyl-glycyl-L-proline methylester product (22.5 g, 78.5 m mole, yield: 95%).
  • N-t-Butyloxycarbonyl-glycyl-L-proline methylester (21.3 g, 74.4 m mole) and 4N hydrogen chloride dioxan solution (150 ml) were mixed, and shaked to dissolve the solid material. It was stirred for 1 hour at room temperature. The solvent was well-distilled off under reduced pressure to obtain the solid glycyl-L-proline methylester hydrochloride (16.4 g, 73.9 m mole, yield: 99%).
  • Glycyl-L-proline methylester hydrochloride (16.0 g, 72.1 m mole) was dissolved in methanol (150 ml), and Indan-2-on (4.75 g, 36.0 m mole) and triethylamine (1.8 g, 18.0 m mole) were added thereto.
  • Sodium syanoborohydride (2.27 g, 36.0 m mole) was gradually added thereto while cooling with ice and stirring. The mixture was stirred for 1 hour while cooling with ice and next for 5 hours at room temperature. A little insoluble material was separated by filtration, and the filtrate was concentrated under reduced pressure.
  • the aqueous layer was removed and the ethyl ether layer was washed with an aqueous solution saturated with sodium chloride.
  • water 150 ml
  • the solution was adjusted to pH 1.5 with 6N hydrochloric acid while stirring and ethyl ether was removed.
  • the water layer was washed with ethyl ether (100 ml) and to the water layer chloroform (200 ml) was added.
  • the solution was adjusted to pH 8.0 with sodium bicarbonate while stirring.
  • the aqueous layer was removed, and chloroform layer was dried with anhydrous sodium sulfate, and the sodium sulfate was separated by filtration.
  • N-(2-indanyl) glycyl-L-proline methylester hydrochloride (4.33 g, 12.8 m mole), N-t-butyloxycarbonyl-L-alanine (2.42 g, 12.8 m mole) and HOBt (1.73 g, 12.8 m mole) were dissolved in chloroform (50 ml), and triethylamine (1.29 g, 12.8 m mole) was added thereto while cooling to -15° C. and then WSC hydrochloride (2.45 g, 12.8 m mole) was gradually added thereto under cooling with ice. Further, the reaction was carried out for 2 hours on ice and next overnight at room temperature.
  • the reaction solution was washed with 10% citric acid, aqueous sodium chloride, 5% sodium bicarbonate, and aqueous sodium chloride in order, and dried with anhydrous sodium sulfate.
  • the solution was concentrated under reduced pressure to obtain the syrupy material (3.88 g). It was further purified by column chromatography (silica gel, developing solvent, chloroform and next ethyl acetate) to obtain N-t-butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline methylester (2.42 g, yield: 64%).
  • N-(2-indanyl) glycyl-L-proline methylester hydrochloride (2.08 g) was recovered from the 10% citric acid solution which was used for washing the above mentioned reaction mixture.
  • N-t-Butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline methylester (2.24 g, 4.7 m mole) was dissolved in 4.8N hydrogen chloride dioxane solution and stirred for 1 hour at room temperature. The solution was distilled off under reduced pressure leaving a residue and diethyl ether was added to the residue to obtain L-alanyl-N-(2-indanyl) glycyl-L-prolyl methylester hydrochloride crystals (1.63 g, yield: 84.1%).
  • N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl) glycyl-L-proline methylester (0.58 g, 0.9 m mole) was dissolved in acetone (3 ml), and a sodium hydroxide solution (0.11 g, 2.8 m mole) in water (3 ml) was added thereto, while cooling in the ice bath. The mixture was stirred for 1 hour. To this solution water (100 ml) was added and the mixture was washed with ethyl ether (50 ml).
  • the ether-washed mixture was neutralized with 1N hydrochloric acid and the desired product was extracted with ethyl acetate.
  • the ethyl acetate layer was dried with anhydrous sodium sulfate and the solvent was distilled off under reduced pressure to obtain a sticky N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl) glycl-L-proline (0.53 g, yield: 93.5%).
  • N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl) glycyl-L-proline (0.53 g, 0.9 m mole) was dissolved in ethanol (4 ml) and the aqueous solution produced by dissolving L-arginine (0.17 g, 0.9 m mole) in water (4 ml) was added thereto. The solution was distilled under reduced pressure leaving a residue and water was added thereto.
  • N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl) glycyl-L-proline L-arginine salt product (0.64 g, yield: 94.5%) was obtained.
  • Dibenzylphosphoryl-L-alanyl-N-(2-indanyl)-glycyl-L-proline (0.77 g, 1.2 m mole) was dissolved in ethanol (4 ml), and the aqueous solution produced by dissolving L-arginine (0.25 g, 1.2 m mole) in water (4 ml), was added thereto. The solvent was distilled off under reduced pressure, and then water was added thereto. This water solution was freeze-dried to obtain dibenzylphosphoryl-L-alanyl-N-(2-indanyl) glycyl-L-proline L-arginine salt (0.84 g, yield: 85.2%).
  • Dibenzylphosphoryl-L-methionyl-L-prolyl-L-proline methylester (1.87 g, 3.0 m mole) was dissolved in acetone (18 ml), and 1N sodium hydroxide (4.5 ml) was added thereto under cooling and the mixture was stirred for 4 hours.
  • water 100 ml was added, and the solution was washed with diethyl ether.
  • Ethyl acetate (200 ml) was added thereto and the mixture was adjusted to pH 2 with 1N hydrochloric acid while stirring strongly. The ethyl acetate phase was separated and dried over anhydrous sodium sulfate.
  • N-(O-Benzyl-P-phenethylphosphonyl)-methionyl-L-prolyl-L-proline benzylester (1.195 g, 1.73 m mole) was dissolved in ethanol (15 ml), and sodium bicarbonate (0.290 g, 3.46 m mole) in water (5 ml) solution was added thereto. Hydrogen gas was passed through the above solution for 6 hours at room temperature and atmospheric pressure in the presence of 30% palladium barium carbonate as a catalyst. The catalyst was filtered off and from the filtrate the solvent was evaporated.
  • N-(O-Benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-proline benzylester (1.195 g, 1.73 m mole) was dissolved in acetone (10 ml), and 1N sodium hydroxide (2.6 ml) was added thereto under cooling in the ice bath. The mixture was stirred for 5.5 hours. To the solution water (100 ml) was added and the mixture was washed with diethyl ether, and then ethyl acetate (200 ml) was added thereto. The solution was adjusted to pH 2 with 1N hydrochloric acid while stirring vigorously.
  • N.sup. ⁇ -benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester hydrochloride (3.26 g, 5.4 m mole) was dissolved in methylene chloride (15 ml), and triethylamine (1.73 g, 17.1 m mole) was added thereto.
  • N.sup. ⁇ -(O-Benzyl-P-phenethylphosphonyl)-N.sup. ⁇ -benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester (1.21 g, 1.5 m mole) was dissolved in methanol (18 ml), and sodium bicarbonate (0.247 g, 2.9 m mole) water (5 ml) solution was added thereto. Hydrogen gas was passed through the solution for 3 hours at room temperature and atmospheric pressure in the presence of 5% palladium carbon as a catalyst. The catalyst was removed by filtration. The solvent was evaporated and the residue was dissolved in water. The solution was freeze-dried to obtain N.sup. ⁇ -phenethylphosphonyl-L-lysyl-L-prolyl-L-proline 2 sodium salt (0.84 g which is yielded quantitatively).
  • Diethyl phosphite (27.64 g, 200 m mole) was dissolved in DMF (200 ml), and to the solution at nitrogen atmosphere while stirring and -15° C., sodium hydride (60% in oil, 9.6 g, 220 m mole) was added. The mixture was stirred for 1 hour at not more than 0° C.
  • phenethyl bromide (37.0 g, 200 m mole) was dropwise added and further the mixture was stirred overnight at room temperature.
  • the DMF solution was distilled under reduced pressure leaving a residue, and the residue was dissolved in ethyl acetate.
  • the ethyl acetate solution was washed with water, and dried with anhydrous sodium sulfate. The solvent was evaporated and the thus obtained oily product was distilled under reduced pressure to obtain diethylphenethylphosphonate (25.0 g, yield: 51.6%) of boiling point 136.5° ⁇ 137.5° C./3 mmH
  • N.sup. ⁇ -Benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester hydrochloride (3.0 g, 5.0 m mole) was dissolved in methylene chloride (15 ml), and triethylamine (1.59 g, 15.7 m mole) was added thereto.
  • N.sup. ⁇ -(O-Ethyl-P-phenethylphosphonyl)-N.sup. ⁇ -benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester (1.46 g, 1.9 m mole) was dissolved in ethanol (10 ml), and L-arginine (0.40 g, 1.9 m mole) in water (5 ml) solution was added thereto. Hydrogen gas was passed through the solution for 5 hours at atmospheric pressure and room temperature in the presence of 5% palladium carbon as a catalyst. The catalyst was removed by filtration and the solvent was evaporated.
  • Dibenzyl n-amylphosphonate (2.74 g, 8.2 m mole) was dissolved in carbon tetrachloride (5 ml), and phosphorus pentachloride (1.80 g, 8.6 m mole) was added to the solution while cooling in the ice bath, and the mixture was stirred for 30 minutes.
  • the mixture was heated to 70° C. at the rate of 10° C./15 minutes, and further stirred for 30 minutes at 70° C.
  • the solution was distilled under reduced pressure in the rotary evaporator to eliminate the solvent, phosphorus oxychloride and benzylchloride.
  • N-(O-Benzyl-P-n-amylphosphonyl)-L-alanyl-L-prolyl-L-proline benzylester (1.26 g, 2.1 m mole) was dissolved in ethanol (20 ml), and sodium bicarbonate (0.354 g, 4.2 m mole) in water (20 ml) solution was added thereto. Hydrogen gas was passed through the solution at room temperature and atmospheric pressure for 3 hours in the presence of 5% palladium carbon as a catalyst. The catalyst was removed by filtration, and the solvent was evaporated.
  • Diethyl phosphite (6.9 g, 50 m mole) was added dropwise to the Grignard reagent solution produced from phenethylbromide (27.5 g, 148 m mole) and magnesium (3.6 g, 148 m mole) in anhydrous diethyl ether (100 ml) while cooling in the ice bath. The mixture was refluxed by heating overnight, and then the remaining Grignard reagent was decomposed by the addition of 1N hydrochloric acid (100 ml) and the diethyl ether was evaporated. Bromine was added to the solution while stirring in the ice bath to give a faintly brown coloured solution. The desired product was extracted with chloroform from the solution.
  • the chloroform layer was dried with anhydrous sodium sulfate, and the chloroform was distilled off under reduced pressure. The thus obtained residue was dissolved in hot ethanol, and the solution was cooled to obtain diphenyl phosphinic acid (10.01 g, yield: 73.0%).
  • N-Diphenethylphosphinyl-L-alanyl-L-prolyl-L-proline benzylester (0.77 g, 1.2 m mole) was dissolved in methanol (20 ml), and sodium bicarbonate (0.103 g, 1.2 m mole) in water (10 ml) solution was added thereto. Hydrogen gas was passed through the above solution at room temperature and atmospheric pressure for 3 hours in the presence of 5% palladium carbon as a catalyst. The catalyst was removed by filtration, and the solvent was evaporated.
  • N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline methylester L-Alanyl-L-prolyl-L-proline methylester hydrochloride (3.3 g, 10.0 mmole) was dissolved in methylene chloride (30 ml), and thiethylamine (3.03 g, 30.0 mmole) was added thereto.
  • N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline methylester (1.21 g, 2.2 mmole) was dissolved in ethanol (20 ml), and sodium bicarbonate (0.18 g, 2.2 mmole) in water (5 ml) solution was added thereto. Hydrogen gas was passed through the above solution for 90 minutes at room temperature and atmospheric pressure in the presence of 5% palladium carbon as a catalyst. The catalyst was removed by filtration, and the solvent was distilled off.
  • PE-300 As an instrument for measuring the blood pressure there was used Programmed Electro-Sphygmomanometer PE-300 (Narco Co., U.S.A.), and the blood pressure was indirectly measured on conscious rats.
  • aqueous solution or suspension of a sample (35 mg/kg) was once force-fed into the stomach by means of a peroral probe.
  • Dry packed capsules containing 50 mg/capsule of an active ingredient were prepared.
  • Tablets for combined use containing a diuretic such as hydrochlorothiazide were prepared by incorporating therein dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline-L-arginate (7.5 mg), hydrochlorothiazide (50 mg), pregelatinized starch (82 mg), microcrystalline cellulose (82 mg), and magnesium stearate (1 mg).
  • a diuretic such as hydrochlorothiazide
  • amino acid derivatives of the present invention are useful as an antihypertensive drug or an intermediate for preparation thereof.

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JP57009341A JPS58126896A (ja) 1982-01-23 1982-01-23 アラニルプロリン誘導体およびこれを含有する降圧剤
JP57-9341 1982-01-23
JP57141831A JPS5931788A (ja) 1982-08-16 1982-08-16 アミノ酸誘導体およびこれを含有する降圧剤
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Cited By (5)

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US4590178A (en) * 1982-01-23 1986-05-20 Ajinomoto Company, Incorporated Amino acid derivatives, methods of preparing said derivatives and antihypertensive drugs containing them
US4595675A (en) * 1979-09-19 1986-06-17 Hoechst Aktiengesellschaft Bicyclic α-iminocarboxylic acid compounds having hypotensive activity
US5389612A (en) * 1989-11-15 1995-02-14 Commissariat A L'energie Atomique Phosphonic peptide inhibitors of bacterial collagenases
US6075121A (en) * 1990-05-15 2000-06-13 Chiron Corporation Modified peptide and peptide libraries with protease resistance, derivatives thereof and methods of producing and screening such
US10053505B2 (en) 2011-01-31 2018-08-21 Kai Yuan Xu Sodium pump antibody agonists and methods of treating heart disease using the same

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JPS60193994A (ja) * 1984-03-13 1985-10-02 Ajinomoto Co Inc Ν−ホスフイノイル化方法
JPH0655758B2 (ja) * 1984-08-24 1994-07-27 味の素株式会社 アミノ酸誘導体
AU595309B2 (en) * 1986-05-09 1990-03-29 Dainippon Pharmaceutical Co. Ltd. Tripeptide derivatives
EP0376040A3 (en) * 1988-12-27 1990-09-12 American Cyanamid Company N-phosphinyl di-and tripeptides as renin inhibitors
IE66205B1 (en) * 1990-06-14 1995-12-13 Paul A Bartlett Polypeptide analogs
US5189023A (en) * 1990-09-04 1993-02-23 American Cyanamid Company Renin inhibitors
FR2689764B1 (fr) * 1992-04-10 1995-05-12 Commissariat Energie Atomique Utilisation de dérivés de peptides pour la fabrication de médicaments inhibiteurs des endopeptidases 24.15 et 24.16.
WO2009037416A1 (en) * 2007-09-20 2009-03-26 Npil Pharmaceuticals (Uk) Limited Process for preparing sulphonamides

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US4316896A (en) * 1978-09-07 1982-02-23 Merck & Co., Inc. Aminoacid derivatives as antihypertensives
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US4379146A (en) * 1981-02-17 1983-04-05 Merck & Co., Inc. Substituted phosphonamides as antihypertensives
US4466913A (en) * 1981-06-30 1984-08-21 Maiji Seika Kaisha Ltd. Phosphorus-containing compounds and process for producing the same
US4432972A (en) * 1981-08-03 1984-02-21 E. R. Squibb & Sons, Inc. Phosphonamidate compounds
US4432971A (en) * 1981-08-03 1984-02-21 E. R. Squibb & Sons, Inc. Phosphonamidate compounds
DE3381378D1 (de) * 1982-01-23 1990-05-03 Ajinomoto Kk Derivate von aminosaeuren und antihypertensive arzneimittel diese enthaltend.

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4595675A (en) * 1979-09-19 1986-06-17 Hoechst Aktiengesellschaft Bicyclic α-iminocarboxylic acid compounds having hypotensive activity
US4590178A (en) * 1982-01-23 1986-05-20 Ajinomoto Company, Incorporated Amino acid derivatives, methods of preparing said derivatives and antihypertensive drugs containing them
US5389612A (en) * 1989-11-15 1995-02-14 Commissariat A L'energie Atomique Phosphonic peptide inhibitors of bacterial collagenases
US6075121A (en) * 1990-05-15 2000-06-13 Chiron Corporation Modified peptide and peptide libraries with protease resistance, derivatives thereof and methods of producing and screening such
US10053505B2 (en) 2011-01-31 2018-08-21 Kai Yuan Xu Sodium pump antibody agonists and methods of treating heart disease using the same
US10214583B2 (en) 2011-01-31 2019-02-26 Kai Yuan Xu Sodium pump antibody agonists and methods of treating heart disease using the same

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EP0085488B1 (en) 1990-03-28
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ES524285A0 (es) 1984-11-16
ES8501412A1 (es) 1984-11-16
ES8501411A1 (es) 1984-11-16
DE3381378D1 (de) 1990-05-03
ES524284A0 (es) 1984-11-16
EP0085488A3 (en) 1985-12-27
US4536395A (en) 1985-08-20

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